Bioengineering bacterially derived immunomodulants: a novel therapeutic approach to IBD Project Summary The long term goal is to develop novel, effective therapeutics that harness the immunomodulatory properties of bacterial molecules for the treatment of inflammatory bowel disease (IBD). The proposal aims to exploit the evolved ability of intestinal pathogens to control inflammatory related signaling pathways in their host, by adapting bacterial effector molecules as therapeutics. A major challenge in realizing the therapeutic potential of these molecules is the ability to engineer a delivery system capable of delivering protein inside intestinal epithelial cells. The objective of this proposal is to create bacterial protein nanoparticles with the ability to deliver bacterial effector proteins, suppress epithelial inflammation, and attenuate the symptoms of IBD. Key outcomes are: (1) a new, long-needed IBD therapeutic that arrests inflammation at the source; (2) a new therapeutic paradigm that utilizes bacterial immunoregulatory mechanisms and engineers a nanoparticle delivery strategy essential for clinical viability. Three specific aims have been set: Aim 1. Engineer the cellular uptake and trafficking properties of nanoparticles to maximize protein delivery. Bacterial protein nanoparticles will be fabricated with different physiochemical features including size, crosslinking density, targeting ligands, and endosomal escape motifs. Epithelial cells exposed to nanoparticles in vitro will be assessed for particle internalization, endosomal escape, and disassociation into soluble protein. Aim 2. Assess in vitro and exvivo biological response to bacterial protein nanoparticles. Various nanoparticle formulations, including those from Aim 1 as well as particles optimized in this aim for transport through mucus, will be applied to unpolarized and polarized epithelial cells, and ex vivo mucosal preparations at a range of dosages. Functional activity will be measured by mucosal uptake, suppression of immune signaling pathways, and reduced levels of inflammatory cytokines. Aim 3. Determine the therapeutic effect of protein nanoparticles optimized for in vivo delivery on diseased animals. Nanoparticles will be modified via surface coatings to increase their ability to traverse the gastrointestinal tract and target inflamed mucosa. These particles will be administered to mice with induced chemical, immunological and genetic models of colitis and to healthy controls. Clinical parameters, mucosal and systemic inflammatory markers, and histopathology will be tracked over relevant time points.